【作者】 商海红；

【导师】 李付广；

【作者基本信息】 中国农业科学院 ， 作物遗传育种， 2009， 博士

【摘要】 农杆菌介导法可以高效的将T-DNA插入棉花基因组中并能稳定遗传,在棉花的遗传转化中应用最为广泛。但农杆菌介导的棉花遗传转化必须以高效的棉花再生体系为基础,胚性愈伤组织诱导困难、非胚性愈伤组织大量发生以及畸形胚状体(畸形苗)高比率发生等问题制约着棉花遗传转化效率的提高。有鉴于此,本研究以绿色荧光蛋白(Enhanced Green Fluorescent Protein,EGFP)为报告基因确定转化体,利用扫描电镜、透射电镜等技术,系统的研究了农杆菌介导的棉花遗传转化过程中不同阶段和不同类型愈伤组织的细胞学和组织学差异,主要结果如下:1)EGFP基因在愈伤组织、胚性愈伤组织、非胚性愈伤组织和体细胞胚均可以表达。利用荧光显微镜可以快速无伤性的鉴定出阳性转化体。2)胚性愈伤组织表面的细胞以细胞团方式存在,这些细胞团由半球形、形状规则、体积相对一致的细胞构成,细胞之间结合紧密,细胞团之间可见间隙;组织学水平上,细胞形态相对一致,表面可见胚性细胞团;细胞超微结构水平上,细胞核大,核仁明显;细胞质电子密度高,富含各种细胞器;质体呈现原质体状态,位于细胞核附近;粗面内质网较为丰富;存在大小不等的液泡。非胚性愈伤组织表面细胞的排列疏松,以单个细胞或者数个细胞构成的简单细胞结合体存在,细胞的形态和体积差别较大;细胞超微结构也有类似之处,有中央大液泡的存在,细胞质被挤压在四周;质地较硬的叶绿体内膜系统发育良好,疯长型的非胚性愈伤组织叶绿体呈类原质体状态,中间形成空泡结构。3)叶绿体在陆地棉体细胞胚胎发生和形态建成过程中发挥着重要的作用。在愈伤组织细胞中,叶绿体以造粉体的状态;随着胚性愈伤组织被诱导出来,以原质体状态存在;待到形成胚状体以后,叶绿体内膜系统逐渐出现;到子叶期的胚叶绿体内膜系统逐渐恢复;再生苗阶段,叶绿体内膜系统恢复正常。研究还发现,叶绿体的异常发育与非胚性愈伤组织发生关系密切,玻璃化胚状体和白化胚状体的叶绿体也存在结构异常。4)诱导出胚性愈伤组织时,细胞外基质就可见;随着胚性愈伤表面形成有数个胚性细胞构成的集合体,细胞外基质发生达到了最多,在愈伤表面形成网络状结构;随着胚性细胞团的发生和形成体细胞胚,细胞外基质逐渐消退。更多还原

【Abstract】 Because of the high transformation efficiency and genetic stability of foreign gene, Agrobacterium-mediated transformation method is considered as the most common technique which has been applied to cotton genetic transformation. However, it requires an efficient plant regeneration procedure. The high genotype-dependence of embryogenic potential, the difficulty embryogenic callus induction, and the high ratio of abnormal somatic embryos restrict the application of biotechnology on cotton development and fundamental research. In view of this, we studied histological structure and ultrastructure of different type callus during the course of Agrobacterium-mediated cotton genetic transformation systematically via such techniques as scanning electron microscope (SEM), transmission electron microscope (TEM) and so on. In this study, enhanced foreign green fluorescent protein (EGFP) was served as the reporter gene to identify the transformant.1) Expression of EGFP was observed in callus, embryogenic callus, non-embryogenic callus and somatic embryo. As EGFP is a stable, cell-autonomous fluorescent protein, positive transformant could be identified by fluorescence microscope non-destructively easily.2) On surface of embryogenic callus, series of nodule-like structures developed, which was formed by small, tightly packed and hemispherical cells. Results of the histological observation revealed that early embryogenic callus displayed similar cellular morphology and embryogenic callus could develop embryonic nodule-like structure through further sub-culturation. Organelles of embryogenic callus cells were located near the nucleus and contained dense cytoplasm. Plastid was degraded into proplastid-like structures with some starch grains. Rough surfaced endoplasmic reticulum (RER) and a series of small vacuoles could also be observed.In contrast, non-embryogenic callus was characterized by high variant of cell morphology and loose cell organization on the surface. The nucleus was located in a narrow strip of cytoplasm between cell wall and the large vacuole which is located in the center. Chloroplasts of hard callus developed well organized membrane system and chloroplasts in cell of overgrown callus were degraded into proplastids which exhibited some vacuole-like structures.3)In the cells of hypocotyls, chloroplasts had abnormal structure. In the cell of callus, plastids were degraded into amyloplast. In the cell of embryogenic callus, chloroplasts were degraded into plastids. When somatic embryo formed, endomembrane system of chloroplast emerged. Once somatic embryo developed into cotyledonary embryos, endomembrane system of chloroplast developed grana lamella and starch grains accumulated in chloroplast stroma. Abnormal development of chloroplast (plastid) was related with the occurrence of non-embryogenic callus closely and there were structural abnormalities of chloroplasts in cells of albino and vitrified somatic embryo.4)As while embryogenic callus were induced, fine fibrils of extracellular matrix(ECM) could be observed. The structural arrangement of the ECM changed during culture. During the formation of a series of multi-cellular embryonic clusters, the ECM was the most abundant and was arranged as network-like layer connected by coarse strands outside of callus. While nodule-like structures formed, ECM was disturbed gradually, and while embryo formed,the ECM network disappeared completely. The ECM was not observed on the surfaces of callus and non-embryogenic callus.更多还原